The present invention relates to pharmaceutical compositions for use in the treatment of disorders or abnormalities of the skin and other body surfaces by photochemotherapy.
Abnormalities or disorders, such as neoplasms or psoriatic plaques, of the skin or other epithelial, e.g. mucosal, organs are conventionally treated by surgery, radiotherapy, cryotherapy or chemotherapy. These treatments however, often have significant and serious drawbacks such as toxicity, carcinogenicity, or other unpleasant side effects or general discomfort resulting from the treatment.
Photochemotherapy or photodynamic therapy (PDT) as it is also known, is a recently up-coming technique for the treatment of various abnormalities or disorders of the skin or other epithelial organs, especially cancers or pre-cancerous lesions, as well as certain non-malignant lesions for example skin complaints such as psoriasis. Photochemotherapy involves the application of photosensitizing (photochemotherapeutic) agents to the affected area of the body or systemic application, followed by exposure to photoactivating light in order to activate the photosensitizing agents and convert them into cytotoxic form, whereby the affected cells are killed or their proliferative potential diminished.
A range of photosensitizing agents are known, including notably the psoralens, the porphyrins, the chlorins and the phthalocyanins. Such drugs become toxic when exposed to light.
Photosensitizing drugs may exert their effects by a variety of mechanisms, directly or indirectly. Thus for example, certain photosensitizers become directly toxic when activated by light, whereas others act to generate toxic species, e.g. oxidising agents such as singlet oxygen or other oxygen-derived free radicals, which are extremely destructive to cellular material and biomolecules such as lipids, proteins and nucleic acids. Psoralens are an example of directly acting photosensitizers; upon exposure to light they form adducts and cross-links between the two strands of DNA molecules, thereby inhibiting DNA synthesis. The unfortunate risk with this therapy is that unwanted mutagenic and carcinogenic side effects may occur.
This disadvantage may be avoided by selecting photosensitizers with an alternative, indirect mode of action. For example porphyrins, which act indirectly by generation of toxic oxygen species, have no mutagenic side effects and represent more favourable candidates for photochemotherapy. Porphyrins are naturally occurring precursors in the synthesis of heme. In particular, heme is produced when iron (Fe3+) is incorporated in protoporphyrin IX (Pp) by the action of the enzyme ferrochelatase. Pp is an extremely potent photosensitizer, whereas heme has no photosensitizing effect.
One such porphyrin-based drug, PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer (Gomer and Dougherty, Cancer Research, 39, p146-151, 1979; originally named Photofrin II) has recently been approved as a photosensitizer in the therapy of certain cancers. PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer consists of large oligomers of porphyrin and it does not readily penetrate the skin when applied topically and must therefore be administered systemically. Thus, its main disadvantage is that since it must be administered parenterally, generally intravenously, it causes photosensitization of the skin which may last for several weeks following i.v. injection. Similar problems exist with other porphyrin-based photosensitizers such as the so-called xe2x80x9chematoporphyrin derivativexe2x80x9d (Hpd) (Lipson et al., J. Natl. Cancer Ins., 60, p1-10, 1961) which has also been reported for use in cancer photochemotherapy (see for example S. Dougherty., J. Natl. Cancer Ins., 52, p1333, 1974; Kelly and Snell, J. Urol., 115, p150, 1976). Hpd is a complex mixture obtained by treating haematoporphyrin with acetic and sulphuric acids, after which the acetylated product is dissolved with alkali. Clearly, there are disadvantages in using an undefined mixture as a drug. Moreover since Hpd must also be administered by injection, it suffers from the same type of undesirable photosensitization drawback as does PHOTOFRIN(copyright)vascular stroma-localizing photosensitizer.
To overcome these problems, precursors of Pp have been investigated for photochemotherapeutic potential. In particular the Pp precursor 5-aminolevulinic acid (ALA) has been investigated as a photochemotherapeutic agent for certain skin cancers. ALA, which is formed from succinyl CoA and glycine in the first step of heme synthesis, is to a limited extent able to penetrate the skin and lead to a localised build-up of Pp; since the action of ferrochelatase (the metallating enzyme) is the rate limiting step in heme synthesis, an excess of ALA leads to accumulation of Pp, the photosensitizing agent. Thus, by applying ALA topically to skin tumours, and then after several hours exposing the tumours to light, a beneficial photochemotherapeutic effect may be obtained (see for example WO91/01727). Since the skin covering basaliomas and squamous cell carcinomas is more readily penetrated by ALA than healthy skin, and since the concentration of ferrochelatase is low in skin tumours, it has been found that topical application of ALA leads to a selectively enhanced production of Pp in tumours.
However, whilst the use of ALA represents a significant advance in the art, photochemotherapy with ALA is not always entirely satisfactory. ALA is not able to penetrate all tumours and other tissues with sufficient efficacy to enable treatment of a wide range of tumours or other conditions and ALA also tends to be unstable in pharmaceutical formulations. Some of these problems may be overcome by using ALA derivatives, for example ester derivatives such as ALA-methylester, ALA-ethylester, ALA-propylester, ALA-hexylester, ALA-heptylester and ALA-octylester and salts thereof as described in our co-pending application WO96/28412.
Like ALA, the ester derivatives exert their effects by enhancing production of Pp; upon delivery to the desired site of action hydrolytic enzymes such as esterases present in the target cells break down the esters into the parent ALA, which then enters the haem synthesis pathway and leads to a build-up of Pp. However, the ester derivatives have a number of advantages over ALA itself. Firstly, they are more lipophilic and better able to penetrate skin and other tissues as compared with ALA; the penetration is both deeper and faster. This is an important advantage, especially for topical administration. Secondly, the esters are better enhancers of Pp production than ALA; Pp production levels following administration of the ALA esters are higher than with ALA alone. Thirdly, the ALA esters demonstrate improved selectivity for the target tissue to be treated, namely the Pp production-enhancing effect is localised to the desired target lesion and does not spread to the surrounding tissues. This is especially evident with tumours. Finally, the esters appear to localise better to the target tissue upon administration. This may be especially important for systemic application, since it means that undesirable photosensitization effects, as reported in the literature for other porphyrin-based photosensitizers, may be reduced or avoided.
Whilst such ALA esters represent a considerable advance in the field of photochemotherapy, not all abnormalities or disorders respond to PDT using known methods to prevent tumour growth and thus there is still a need for better and alternative photochemotherapeutic agents to retard or prevent tumour growth. The present invention thus aims to provide photochemotherapeutic compositions which have an enhanced photochemotherapeutic effect over those described in the prior art.
Studies conducted by the authors have shown that efficient eradication of tumours by PDT requires destruction of both cellular components and also vascular stroma of tumours (Peng and Moan, Br. J. Cancer, 72, p565-574, 1995; Peng et al., Cancer Res., 55, p2620-2626, 1995 and Peng et al., Ultrastructural Pathology, 20, p109-129, 1996). ALA has proven utility in treating tumours and the PpIX synthesized endogenously from ALA localizes within tumour cells. Furthermore, locally applied ALA does not cause skin sensitization and has no mutagenic effect on the DNA of cells. Systemically applied ALA shows no sensitization 24 hours after administration. As mentioned previously however, ALA is not able to penetrate all tumours and has only been found to have good efficacy for the treatment of superficial lesions of the skin with a thickness less than 2-3 mm. No good clinical results have been obtained using topically or systemically administered ALA-PDT on thicker skin lesions or thicker lesions of the aerodigestive tract or other internal hollow organs. PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer is known to distribute mainly in vascular stroma of tumours, but as mentioned above, is associated with a prolonged risk of skin photosensitization.
However, it has now surprisingly been found, that the use of a vascular stroma-localizing photosensitizer, e.g. PHOTOFRIN(copyright)vascular stroma-localizing photosensitizer, tetra(meso-hydroxyphenyl)chlorin (m-THPC), chlorin e6; aluminium phthalocyanine di-sulfonate or aluminium phthalocyanine tetra-sulfonate in combination with a protoporphyrin precursor photochemotherapeutic agent, e.g. ALA or its methyl or butyl esters, enhances the efficiency of PDT relative to the use of one of the agents alone. A synergistic effect was observed between the vascular stroma-localizing photosensitizer and the protoporphyrin precursor photochemotherapeutic agent, resulting in improved suppression of tumour growth compared to the expected additive effect of the agents alone. This advantageous, synergistic effect was surprisingly observed even when using the vascular stroma-localizing agent at a less than therapeutic dose (sub-therapeutic) which whilst not effective at reducing tumour growth, reduces or avoids the risk of skin photosensitivity. For example, the growth of tumours treated in this way were found to be reduced by using ALA at a therapeutic dose and PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer (or m-THPC) at a low non-therapeutic level. The reduction in growth was significantly greater when compared to the additive effects of results obtained using ALA at a therapeutic dose or PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer (or m-THPC) at a therapeutic dose. This suggests a hitherto unrecognized synergistic effect between these different types of photochemotherapeutic agents, even at non-therapeutic doses.
The synergistic effect, even at sub-therapeutic levels, has significant clinical implications. Firstly, improved PDT is achieved which is not limited to superficial skin lesions, but may also be used to treat thick skin lesions and superficial lesions of internal hollow organs, and secondly, if sub-therapeutic doses of the vascular stroma-localizing photosensitizer are employed, the skin phototoxicity associated with these agents may be avoided.
In one aspect, the present invention thus provides a pharmaceutical composition for the treatment of disorders or abnormalities of external or internal surfaces of the body which are responsive to photochemotherapy, comprising a protoporphyrin precursor photochemotherapeutic agent together with a vascular stroma-localizing photosensitizer, optionally together with at least one surface penetration assisting agent and optionally with one or more chelating agents. In particular, the therapeutic efficacy of the photochemotherapeutic agents is enhanced, ie. PDT is enhanced relative to the use of one of the agents alone. More particularly, the therapeutic efficacy is synergistically enhanced. In a preferred aspect of the invention, the vascular stroma-localizing photosensitizer is provided at a sub-therapeutic dose.
Alternatively viewed, the invention can be seen to provide the use of a protoporphyrin precursor photochemotherapeutic agent together with a vascular stroma-localizing photosensitizer, optionally together with at least one surface penetration assisting agent and optionally with one or more chelating agents in the preparation of a composition for the treatment of disorders or abnormalities of external or internal surfaces of the body which are responsive to photochemotherapy.
The invention also extends to novel compositions of protoporphyrin precursor photochemotherapeutic agents and vascular stroma-localizing photosensitizers, optionally together with at least one surface penetration assisting agent and optionally with one or more chelating agents.
It will be appreciated that certain vascular stroma-localizing photosensitizers, e.g PHOTOFRIN(copyright)vascular stroma-localizing photosensitizer, can not be administered topically, and thus unless both photochemotherapeutic agents of compositions of the invention are administered parenterally, the administration will be by use of separate preparations either administered at the same time or following one another.
Thus, viewed from a further aspect, the invention thus provides a product comprising a protoporphyrin precursor photochemotherapeutic agent and a vascular stroma-localizing photosensitizer, optionally together with at least one surface-penetration assisting agent, and optionally one or more chelating agents as a combined preparation for simultaneous, separate or sequential use in treating disorders or abnormalities of external or internal surfaces of the body which are responsive to photochemotherapy.
Furthermore, the use of a protoporphyrin precursor photochemotherapeutic agent and a vascular stroma-localizing photosensitizer, optionally together with at least one surface-penetration assisting agent, and optionally one or more chelating agents in the preparation of a product for simultaneous, separate or sequential use in treatment of disorders or abnormalities of external or internal surfaces of the body which are responsive to photochemotherapy, forms a further aspect of the invention.
As used herein, xe2x80x9cprotoporphyrin precursor photochemotherapeutic agentsxe2x80x9d refers to structural precursors of protoporphyrin and derivatives thereof which function as photochemotherapeutic agents, for example ALA, porphobilinogen or precursors or derivatives thereof, which form a preferred aspect of the invention. Generally such agents localize to cells of the lesion, e.g. a tumour or diseased cell.
xe2x80x9cVascular stroma-localizing agentsxe2x80x9d refers to agents which generally localize to the vascular stroma after administration. Suitable vascular stroma-localizing agents include:
HpD;
Hematoporphyrines such as PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer (Quadra Logic Technologies Inc., Vancouver, Canada) and Hematoporphyrin IX (HpIX);
Photosan III (Seehof Laboratorium GmbH, Seehof, Wesselburenerkoog, Germany);
Clorins such as tetra(m-hydroxyphenyl)chlorins (m-THPC) and their bacteriochlorins (Scotia Pharmaceuticals Ltd, Surrey, UK), mono-L-aspartyl chlorin e6 (NPeG) (Nippon Petrochemical Co., CA, USA), chlorin e6 (Porphyrin Products Inc.), benzoporphyrins (Quadra Logic Technologies Inc., Vancouver, Canada)(e.g. benzoporphyrin derivative monoacid ring A, BPD-MA) and purpurines (PDT Pharmaceuticals Inc., CA, USA) (e.g. tin-ethyl etiopurpurin, SnET2);
phthalocyanines (e.g. zinc-(Quadra Logic Technologies Inc., Vancouver, Canada), some aluminium- or silicon phthalocyanines, which may be sulfonated, in particular sulfonated phthalocyanines such as aluminium phthalocyanine di-sulfonate (A1PcS2a) or aluminium phthalocyanine tetra-sulfonate (A1PcS4));
porphycenes;
hypocrellins;
Protoporphyrin IX (PpIX);
Hematoporphyrin di-ethers;
Uroporphyrins;
Coproporphyrins;
Deuteroporphyrin; and
Polyhematoporphyrin (PHP), and precursors and derivatives thereof.
As mentioned previously, PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer comprises a mixture of different components and each of these separate components or combinations thereof may be used to provide the vascular stroma-localizing agent.
xe2x80x9cVascular stromaxe2x80x9d is intended to signify the vascular connective tissue, matrix and its components and nerves in addition to cells such as macrophages and fibroblasts present in the vascular system and other cells which infiltrate into the stroma. It will be appreciated that the region of localization will depend on the time post-administration at which localization is determined. Thus, photosensitizers which initially localize in cells may relocate to the stroma, and vice versa. For example, aluminium phthalocyanine di-sulfonate localizes initially to the stroma whereas 24-72 hours post-injection the majority of the agent is found in cells.
In general however, vascular stroma-localizing agents are considered to be those present in the stroma in the 24 hours following administration. This may however be manipulated by performing PDT at different times post-administration of the agent such that the agent(s) behaves appropriately as a vascular stroma or lesion-localizing agent at the time of irradiation.
Preferably the protoporphyrin precursor is ALA or a precursor or derivative thereof and the vascular stroma-localizing photosensitizer is a Hematoporphyrin (particularly PHOTOFRIN(copyright)vascular stroma-localizing photosensitizer), a chlorin (particularly m-THPC or chlorin e6) or a sulphonated phthalocyanine (particularly aluminium phthalocyanine di-sulfonate or aluminium phthalocyanine tetra-sulfonate).
The term xe2x80x9cprecursorsxe2x80x9d as used herein refers to precursors for the agent which are converted metabolically to that agent and are thus essentially equivalent to that agent, e.g. ALA. Thus the term xe2x80x9cprecursorxe2x80x9d covers biological precursors for protoporphyrin in the metabolic pathway for haem biosynthesis. xe2x80x9cDerivativesxe2x80x9d include pharmaceutically acceptable salts and chemically modified agents, for example esters such as ALA esters as described hereinbefore.
Surface-penetration assisting agents may be used which have a beneficial effect in enhancing the photochemotherapeutic effect. Such agents may be used even when the photochemotherapeutic agents are not administered topically. Dialkylsulphoxides such as dimethylsulphoxide (DMSO) are especially preferred. This is described in detail in WO 95/07077.
The surface-penetration assisting agent may be any of the skin-penetration assisting agents described in the pharmaceutical literature e.g. HPE-101 (available from Hisamitsu), DMSO and other dialkylsulphoxides, in particular n-decylmethyl-sulphoxide (NDMS), dimethylsulphacetamide, dimethylformamide (DMFA), dimethylacetamide, glycols, various pyrrolidone derivatives (Woodford et al., J. Toxicol. Cut. and Ocular Toxicology, 5, p167-177, 1986), and Azone(copyright) (Stoughton et al., Drug Dpv. Ind. Pharm., 9, p725-744, 1983), or mixtures thereof.
DMSO however has a number of beneficial effects and is strongly preferred. Thus, in addition to the surface-penetration assisting effect (DMSO is particularly effective in enhancing the depth of penetration of the active agent into the tissue), DMSO has anti-histamine and anti-inflammatory activities, leading to a reduction in pain during the light exposure process. In addition, DMSO has been found to increase the activity of the enzymes ALA-synthase and ALA-dehydrogenase (the enzymes which, respectively, form and condense ALA to porphobilinogen) thereby enhancing the formation of the active form, Pp.
However, in certain conditions such as psoriasis, the lesions are relatively easily penetrated and the penetrating agent may be less beneficial. In some circumstances, for example in the case of skin cancers where the lesions are difficult to penetrate, the surface penetration assisting agent may be applied in a preliminary step, generally at a higher concentration.
Thus, the various active components need not be applied simultaneously within the same composition, but may, according to clinical need, be administered separately and sequentially. Indeed, it has been observed that in many cases a particularly beneficial photochemotherapeutic effect may be obtained by pre-treatment with the surface-penetration assisting agent in a separate step, prior to administration of the photochemotherapeutic agents. Furthermore, in some situations a pre-treatment with the surface-penetration assisting agent, followed by administration of the photochemotherapeutic agent in conjunction with the surface-penetration assisting agent has been found to be beneficial. When a surface-penetration assisting agent is used in pre-treatment this may be used at high concentrations, e.g. up to 100% (w/w). If such a pre-treatment step is employed, the photochemotherapeutic agent may subsequently be administered up to several hours following pre-treatment e.g. at an interval of 5-60 minutes following pre-treatment.
Malik et al in Proceedings of Photodynamic Therapy of Cancer, 2078, p355-362, 1993, described in vitro studies of the effects of ALA, on induction of protoporphyrin biosynthesis, and subsequent killing by photodestruction, of B16 melanoma cells in culture, which had previously been incubated with DMSO as differentiation inducer and/or allyl-isopropyl-acetamide as porphyrogenic agent, to increase endogenous porphyrin levels prior to incubation with the ALA.
Doodstar et al in Biochemical Pharmacology, 42(6), p1307-1303, 1991, describe an investigation into the effects of culture conditions on hepatocytes in culture, and in particular the effects of ALA and DMSO, alone or in combination, on increasing the activities of cytochrome P450-dependent mixed function oxidase and UDP-glucuronosyl transferase, by increasing intracellular haem concentrations, in hepatocyte cells in culture.
Chelating agents are optionally contained in the pharmaceutical composition or product of the invention. Such agents may be useful for two effects, firstly to enhance the stability of the protoporphyrin precursor photochemotherapeutic agent, e.g. ALA and secondly to enhance accumulation of Pp. The latter effect is achieved by the chelation of iron, thereby preventing the inactivating action of the enzyme ferrochelatase in incorporating the metal into Pp, leading to Pp build-up. The photosensitizing effect is thus enhanced.
Hanania et al in Cancer Letters, 65, p127-131, 1992 propose the use of ALA in combination with-chelating agents in photochemotherapy of topically treated tumours.
Aminopolycarboxylic acid chelating agents are particularly suitable for use in this regard, including any of the chelants described in the literature for metal detoxification or for the chelation of paramagnetic metal ions in magnetic resonance imaging contrast agents. Particular mention may be made of EDTA, CDTA (cyclohexane diamine tetraacetic acid), DTPA, DOTA and 1,10-phenanthroline. EDTA is preferred, especially for the stabilisation of ALA. To achieve the iron-chelating effect, desferrioxamine and other siderophores may also be used, e.g. in conjunction with aminopolycarboxylic acid chelating agents such as EDTA.
The compositions of the invention or used according to the invention may additionally be formulated and/or administered with other agents, to improve the efficacy of PDT. Thus for example, angiogenesis inhibitors (anti-angiogenic drugs) which have been found to be useful for treating tumours (O""Reilly et al., Nature Medicine, 2, p689-692, 1996; Yamamoto et al., Anticancer Research, 14, p1-4, 1994; and Brooks et al., J. Clin. Invest., 96, p1815-1822, 1995) may be used together with compositions of the invention in PDT to further damage the vascular system of the tumour. Angiogenesis inhibitors which may be used include TNP-470 (AGM-1470, a synthetic analogue of a fungal secretion product called fumagillin; Takeda Chemical Industries Ltd., Osaka, Japan), angiostatin (Surgical Research Lab. at Children""s Hospital Medical Center of Harvard Medical School) and integrin xcex1vxcex23 antagonists (e.g. monoclonal antibody to intefrin xcex1vxcex23, The Scripps Research Institute, LaJolla, Calif.).
Alternatively, or additionally, immunotherapy agents (e.g. antibodies or effectors such as macrophage activating factor) or chemotherapy agents may be used to improve PDT according to the invention. Administration of these supplementary agents should be performed in terms of route, concentration and formulation, according to known methods for using these agents. These additional agents may be administered before, after or during PDT, depending on their function. For example, angiogenesis inhibitors may be added 5-10 days after PDT to prevent tumour regrowth.
Glucose has also been found to assist PDT when applied either topically or systemically. Although not wishing to be bound by theory, it appears that administration of glucose results in a lowering of pH which increases the hydrophobic properties of protoporphyrins such that they can penetrate cells more easily. When topical administration is contemplated, conveniently the formulation, e.g. a cream, may contain 0.01 to 10% glucose (w/w).
A preferred composition or product according to the invention, comprises ALA or a precursor or derivative thereof, PHOTOFRIN(copyright)vascular stroma-localizing photosensitizer, DMSO, EDTA and desferrioxamine.
As mentioned above, a synergistic effect has been observed, between the protoporphyrin precursor and the vascular stroma-localising photochemotherapeutic agent, whereby the efficiency of PDT is enhanced. Thus, this enables sub-therapeutic dosages of the photochemotherapeutic agent to be used ie. dosages which, were the individual photochemotherapeutic-agent to be administered on its own, would not suffice-to achieve a beneficial photochemotherapeutic effect.
It has in particular been found that beneficial results may be obtained using the protoporphyrin precursor agent, preferably ALA or a derivative thereof, at a therapeutic dose range, standard for PDT using such a photochemotherapeutic agent solely, in conjunction with a sub-therapeutic dose of the vascular stroma-localising agent, preferably PHOTOFRIN(copyright)vascular stroma-localizing photosensitizer.
The concentration of the protoporphyrin precursor photochemotherapeutic agent, e.g. ALA in the composition is conveniently in the range 1 to 40%, e.g. 2 to 25, preferably 5 to 20%; the concentration of the vascular stroma-localizing photosensitizer, e.g. PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer is conveniently in the range 0.1 to 1% or m-THPC is conveniently in the range 0.01-10%, the concentration of chelating agent is preferably in the range 1 to 20% e.g. about 2 to 10%, e.g. 2.5%; the concentration of surface penetration assisting agent, e.g. DMSO, is preferably in the range 2 to 50% e.g. about 10%. All percentages stated above are by weight. The concentration of agent which is required depends on the particular agent which is used and clearly should be modified as appropriate according to information and techniques known in the art. Furthermore, it will be appreciated that the concentration used depends on the method of application and on the time for which the composition is applied. However, as mentioned above, where the surface penetration assisting agent is administered separately in a preliminary step, it may be applied at higher concentrations, even up to 100%. Compositions of the invention may be administered exclusively topically (by application to internal or external surfaces using for example a cream, instillation, local internal administration/injection or inhalation) or systemically (e.g. orally or by intravenous injection) or by a combination of these methods in which one or more components of the composition is administered topically and the other components are administered systemically.
The total dosage of the vascular stroma-localizing photosensitizer administered, e.g. by intravenous administration is preferably in the range of 0.01 to 10 mg/kg body weight, for example for PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer preferably 0.01 to 1 mg/kg body weight (sub-therapeutic dose) or for m-THPC preferably 0.01 to 0.2 mg/kg body weight and for the protoporphyrin precursor photochemotherapeutic agent in the range of 1 to 500 mg/kg, e.g. 1 to 250 mg/kg, for example for ALA in the range 1 to 250 mg/kg, preferably 20 to 70 mg/kg body weight.
It will be appreciated that the dosage required depends on the mode and route of administration, the agent employed and the lesion to be treated. Whilst sub-therapeutic doses of the vascular stroma-localizing photochemotherapeutic agent are preferred, this may be increased if for example a large thick lesion or a difficult type of disease (e.g. melanoma) is to be treated. The observed synergistic effect allows the levels of both the vascular stroma-localizing agent and the protoporphyrin precursor agent to be reduced below normal therapeutic levels.
Alternatively viewed, this aspect of the invention also provides a kit for use in photochemotherapy of disorders or abnormalities of external or internal surfaces of the body comprising:
a) a first container containing a protoporphyrin precursor photochemotherapeutic agent, e.g. ALA or a precursor or derivative thereof;
b) a second container containing a vascular stroma-localizing photosensitizer, e.g. PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer or m-THPC; and optionally
c) at least one surface-penetrating agent contained within said first or second container or in a third container; and/or
d) one or more chelating agents contained either within said first, second or third container or in a fourth container;
wherein said first or second container may be absent and the agent or photosensitizer of a) or b) above is present in one of the other containers present in the kit.
Additional components of the kit may also be provided such as angiogenesis inhibitors or glucose as mentioned hereinbefore.
The abnormalities and disorders which may be treated according to the present invention include any malignant, pre-malignant and non-malignant abnormalities or disorders responsive to photochemotherapy e.g. tumours, dysplasia or other growths, non-malignant gynaecological diseases such as menorrhagia, endometriosis and ectopic pregnancy, skin disorders such as psoriasis, actinic keratoses and acne, skin abrasions, and other diseases or infections e.g. bacterial, viral or fungal infections, for example Herpes virus infections. The invention is particularly suited to the treatment of diseases, disorders or abnormalities where discrete lesions are formed (lesions is used here in a broad sense to include tumours and the like). However, the methods of the invention may also be used to treat abnormalities and disorders not characterized by a lesion but displaying discrete separable entities which characterize that disease, for example, abnormalities in the blood or bone marrow indicative of diseases of said blood or marrow or indicative of a disease or disorder located elsewhere in the body which additionally may result in the presence of abnormalities in the blood or marrow, e.g circulating transformed cells.
The internal and external body surfaces which may be treated according to the invention include the skin and all other epithelial and serosal surfaces, including for example mucosa, the linings of organs e.g. the respiratory, gastro-intestinal and genito-urinary tracts, and glands with ducts which empty onto such surfaces (e.g. liver, sebaceous glands, mammary glands, salivary glands and seminal vesicles). In addition to the skin, such surfaces include for example the lining of the vagina, the endometrium and the urothelium. Such surfaces may also include cavities formed in the body following excision of diseased or cancerous tissue e.g. brain cavities following the excision of tumours such as gliomas.
Exemplary surfaces thus include: (i) skin and conjunctiva; (ii) the lining of the mouth, pharynx, oesophagus, stomach, intestines and intestinal appendages, rectum, and anal canal; (iii) the lining of the nasal passages, nasal sinuses, nasopharynx, trachea, bronchi, and bronchioles; (iv) the lining of the ureters, urinary bladder, and urethra; (v) the lining of the vagina, uterine cervix, and uterus; (vi) the parietal and visceral pleura; (vii) the lining of the peritoneal and pelvic cavities, and the surface of the organs contained within those cavities; (viii) the dura mater and meninges; (ix) any tumours in solid tissues that can be made accessible to photoactivating light e.g. either directly, at time of surgery, or via an optical fibre inserted through a needle.
The compositions of the invention may be formulated in conventional manner optionally with one or more physiologically acceptable carriers or excipients, according to techniques well known in the art. Topical compositions are preferred except when a single composition according to the invention is prepared and a topical composition is not suitable for administration of an agent, e.g. PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer in which case systemic application, at least of that agent, will be necessary. Topical compositions include gels, creams, ointments, sprays, lotions, salves, sticks, soaps, powders, pessaries, aerosols, drops and any of the other conventional pharmaceutical forms in the art.
Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will, in general, also contain one or more emulsifying, dispersing, suspending, thickening or colouring agents. Powders may be formed with the aid of any suitable powder base. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing, solubilising or suspending agents. Aerosol sprays are conveniently delivered from pressurised packs, with the use of a suitable propellant.
Alternatively, the surface penetration assisting agent is applied topically in a separate step, and the vascular stroma-localizing photosensitizer, e.g. PHOTOFRIN(copyright) vascular stroma-localizing photosensitizer and protoporphyrin precursor photochemotherapeutic agent, e.g. ALA, optionally together or separately with one or more chelating agents may be administered by an alternative route e.g. orally or parenterally for example by intradermal, subcutaneous, intraperitoneal or intravenous injection. Alternative pharmaceutical forms thus include plain or coated tablets, capsules, suspensions and solutions containing the active components optionally together with one or more inert conventional carriers and/or diluents, e.g. with corn starch, lactose, sucrose, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, citric acid, tartaric acid, water, water/ethanol, water/glycerol, water/sorbitol, water/polyethyleneglycol, propyleneglycol, stearylalcohol, carboxymethylcellulose or fatty substances such as hard fat or suitable mixtures thereof.
Following administration to the surface or systemic administration, or both, the area treated is exposed to light to achieve the photo-chemotherapeutic effect. This can generally be in the order of a few minutes to 96 hours, preferably 15 minutes to 3 hours. The length of time before light administration is also dependant on the mode of administration, and also the dose and particular agent employed.
The irradiation will in general be applied at a dose level of 10 to 250 Joules/cm2 with an intensity of 20-200 mW/cm2 when a laser is used or a dose of 10-540 J/cm2 with-an intensity of 50-300 mW/cm2 when a lamp is applied. At 100 Joules/cm2, penetration of the radiation is found to be relatively deep. Irradiation is preferably performed for 5 to 30 minutes, preferably for 15 minutes. A single irradiation may be used or alternatively a light split dose in which the light dose is delivered in two fractions, e.g. a few minutes to a few hours between irradiations, may be used.
The wavelength of light used for irradiation may be selected to achieve a more efficacious photochemotherapeutic effect. Conventionally, when porphyrins are used in photochemotherapy they are irradiated with light at about the absorption maximum of the porphyrin. Thus, for example in the case of the prior art use of ALA in photochemotherapy of skin cancer, wavelengths in the region 350-640 nm, preferably 610-635 nm were employed. However, by selecting a broad range of wavelengths for irradiation, extending beyond the absorption maximum of the porphyrin, the photosensitizing effect may be enhanced. Whilst not wishing to be bound by theory, this is thought to be due to the fact that when Pp, and other porphyrins, are exposed to light having wavelengths within its absorption spectrum, it is degraded into various photo-products including in particular photoprotoporphyrin (PPp). PPp is a chlorin and has a considerable photo-sensitizing effect; its absorption spectrum stretches out to longer wavelengths beyond the wavelengths at which Pp absorbs ie. up to almost 700 nm (Pp absorbs almost no light above 650 nm). Other agents have been identified for use in PDT which absorb light of even higher wavelengths. Thus in conventional photochemotherapy, the wavelengths used do not excite PPp and hence do not obtain the benefit of its additional photosensitizing effect. Irradiation with wavelengths of light in the range 350-900 nm has been found to be particularly effective although this depends on the agent which is employed. It is particularly important to include the wavelengths between 600 and 700 nm, especially between 630 and 690 nm, specifically the range 630 to 670 nm.
A further aspect of the invention thus provides a method of photochemotherapeutic treatment of disorders or abnormalities of external or internal surfaces of the body, comprising administering to the affected surfaces, a composition or product as hereinbefore defined, and exposing said surfaces to light, preferably to light in the wavelength region 350-900 nm. Alternatively however a light of a narrow wavelength may be used, e.g. when a laser is used, light at a wavelength around 630 nm may be used.
Methods for irradiation of different areas of the body, e.g. by lamps or lasers are well known in the art (see for example Van den Bergh, Chemistry in Britain, May 1986 p. 430-439).
It will be appreciated that the method of therapy using compounds of the invention inevitably involves the fluorescence of the disorder or abnormality to be treated. Whilst the intensity of this fluorescence may be used to eliminate abnormal cells, the localization of the fluorescence may be used to visualize the size, extent and situation of the abnormality or disorder. This is made possible through the ability of the agents used in accordance with the invention to preferentially localize to non-normal tissue.
The abnormality or disorder thus identified or confirmed at the site of investigation may then be treated through alternative therapeutic techniques e.g. surgical or chemical treatment, or by the method of therapy of the invention by continued build up of fluorescence or through further application of compounds of the invention at the appropriate site. It will be appreciated that diagnostic techniques may require lower levels of fluorescence for visualization than used in therapeutic treatments. Thus, generally, concentration ranges of 1 to 50% e.g. 1-5% (w/w) are suitable. Sites, methods and modes of administration have been considered before with regard to the therapeutic uses and are applicable also to diagnostic uses described here. The compounds of the invention may also be used for in vitro and in vivo diagnostic techniques, for example for examination of the cells contained in body fluids. The higher fluorescence associated with non-normal tissue may conveniently be indicative of an abnormality or disorder. This method is highly sensitive and may be used for early detection of abnormalities or disorders, for example bladder or lung carcinoma by examination of the epithelial cells in urine or sputum samples, respectively. Other useful body fluids which may be used for diagnosis in addition to urine and sputum include blood, semen, tears, stools, spinal fluid etc. Tissue samples or preparations may also be evaluated, for example biopsy tissue or bone marrow samples. The present invention thus extends to the use of compounds of the invention, or salts thereof for diagnosis according to the aforementioned methods for photochemotherapy, and products and kits for performing said diagnosis.
A further aspect of the invention relates to a method of in vitro diagnosis, of abnormalities or disorders by assaying a sample of body fluid or tissue of a patient, said method comprising at least the following steps:
i) admixing said body fluid or tissue with a compound as described hereinbefore,
ii) exposing said mixture to light,
iii) ascertaining the level of fluorescence, and
iv) comparing the level of fluorescence to control levels.